Cloning of the Glycoprotein 58 Region of the Guinea Pig Cytomegalovirus Glycoprotein B Gene
Human cytomegalovirus (CMV) can cause deafness, blindness and varying degrees of mental retardation in prenatally infected infants. CMV infections also account for many medical complications such as pneumonia faced by individuals whose immune systems have been weakened by chemotherapy or organ transplants. Development of a vaccine to counter this virus will stem from the discovery of structural proteins that will act effectively as antigens, thus eliciting a humoral response from the human immune system. One CMV envelope protein, glycoprotein B, has been determined to be the major target for virus-neutralizing antibodies. Since guinea pig CMV undergoes transplacental and intrauterine infection of the fetus in a manner similar to humans, guinea pig CMV was used in this experiment as an animal model. Cloning and expression of the carboxy I terminus of guinea pig gB, (identified as gp58) was undertaken as a preliminary step towards future examination of the antigenic potential of gp58. Attempts were made, using polymerase chain reaction and other standard subcloning techniques, to clone gp58 and to ligate it into New England Biolab's pMal-p2 expression vector for eventual expression and purification using the Protein Fusion and Purification System. A PCR generated fragment was successfully ligated into pMal-p2, however it was in the incorrect orientation for proper protein translation. Restriction endonuclease digestion of a plasmid previously engineered to contain the gp58 segment was performed. The resulting fragment was then ligated into the vector. Subsequent analysis of this fragment revealed that the ligated insert was not· the desired gp58 region of the guinea pig cytomegalovirus.
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